Macrophages eat cancer cells using their own calreticulin as a guide

ABSTRACT

Therapeutic and diagnostic methods are provided, which methods relate to the induction of expression of calreticulin on phagocytic cells. Specifically, the methods relate to macrophage-mediated programmed cell removal (PrCR), the methods comprising increasing PrCR by contacting a phagocytic cell with a toll-like receptor (TLR) agonist; or down-regulating PrCR by contacting a phagocytic cell with an inhibitor of Bruton&#39;s tyrosine kinase (BTK). In some embodiments, an activator of TLR signaling or a BTK agonist is provided in combination with CD47 blockade.

CROSS REFERENCE

This application is a 371 application and claims the benefit of PCTApplication No. PCT/US2016/014334, filed Jan. 21, 2016, which claimsbenefit of U.S. Provisional Patent Application No. 62/106,050, filedJan. 21, 2015, which applications are incorporated herein by referencein their entirety.

BACKGROUND

The reticuloendothelial system (RES) is a part of the immune system. TheRES consists of the phagocytic cells located in reticular connectivetissue, primarily monocytes and macrophages. The RES consists of 1)circulating monocytes; 2) resident macrophages in the liver, spleen,lymph nodes, thymus, submucosal tissues of the respiratory andalimentary tracts, bone marrow, and connective tissues; and 3)macrophage-like cells including dendritic cells in lymph nodes,Langerhans cells in skin, and microglial cells in the central nervoussystem. These cells accumulate in lymph nodes and the spleen. The RESfunctions to clear pathogens, particulate matter in circulation, andaged or damaged hematopoietic cells.

To eliminate foreign cells or particles in the innate immune response,macrophage-mediated phagocytosis is induced when the phosphatidylserinereceptor (PSR) reacts to phosphatidylserine (PS), which can beexternalized from the membranes of dead cells, such as apoptotic andnecrotic cells. In turn, the interaction between PS and PSR plays acrucial role in the clearance of apoptotic cells by macrophages. Oncephagocytosis has been performed by macrophages, the inflammatoryresponse is downregulated by an increase in factors such as IL-10,TGF-β, and prostaglandin E2 (PGE2). The strict balance between theinflammatory and anti-inflammatory responses in both innate and adaptiveimmunity plays a critical role in maintaining cellular homeostasis andprotecting a host from extrinsic invasion.

The causal relationship between inflammation and the neoplasticprogression is a concept widely accepted. Data now support the conceptof cancer immunosurveillance—that one of the physiologic functions ofthe immune system is to recognize and destroy transformed cells.However, some tumor cells are capable of evading recognition anddestruction by the immune system. Once tumor cells have escaped, theimmune system may participate in their growth, for example by promotingthe vascularization of tumors.

Both adaptive and innate immune cells participate in the surveillanceand the elimination of tumor cells, but monocytes/macrophages may be thefirst line of defense in tumors, as they colonize rapidly and secretecytokines that attract and activate dendritic cells (DC) and naturalkiller (NK) cells, which in turn can initiate the adaptive immuneresponse against transformed cells.

Malignant cellular transformation occurs through a progression ofgenetic mutations and epigenetic reprogramming that activate oncogenesand inactivate tumor suppressor pathways leading to inheritance ofseveral hallmarks shared by most cancer cells including:self-sufficiency in growth signals, insensitivity to anti-growthsignals, tissue invasion and metastasis, poorly regulated replicativepotential, sustained angiogenesis, and evasion of cell death by avariety of pathways, including apoptosis. In addition to these cellintrinsic properties, recent evidence suggests that many cancers arealso able to evade the immune system through several distinctmechanisms.

Exploration of mechanisms by which cells avoid being cleared byphagocytosis can provide insight into ways for improving transplantationsuccess of hematopoietic and progenitor stem cells, and improved methodsof removing cancer cells from the body. The present invention satisfiesthese, and other, needs.

SUMMARY OF THE INVENTION

Therapeutic and diagnostic methods are provided, which methods relate tomacrophage-mediated programmed cell removal (PrCR). It is shown hereinthat phagocytic cells, e.g. macrophages, in response to TLR signalingupregulate expression of calreticulin (CRT) on the phagocytic cellsurface. The CRT on the surface of the phagocytic cell interacts withtarget cells, e.g. cancer cells, to initiate PrCR. The upregulation ofCRT by the phagocytic cell is shown to involve a Bruton's tyrosinekinase (BTK) signaling pathway, and inhibition of BTK downregulatescalreticulin on the phagocyte cell surface, thereby reducing PrCR. Themethods of the invention increase PrCR by contacting a phagocytic cellwith a TLR agonist; or down-regulate PrCR by contacting a phagocyticcell with an inhibitor of BTK. The contacting can be performed in vitro,e.g. to prime phagocytic cells for therapeutic purposes; or can beperformed in vivo for therapeutic purposes. The expression of CRT on thephagocytic cell surface provides a biomarker for determining thephagocytic capability of the cell.

In one embodiments of the invention, an activator of TLR signaling or aBTK agonist is provided in combination with CD47 blockade, where thePrCr is increased relative to the cell removal in the presence of eitheragent as a monotherapy. In some embodiments, a population of cellscomprising macrophages is contacted in vitro or in vivo with a dose of aTLR agonist or a BTK agonist that is effective in increasing CRT on thecell surface of the macrophage by at least about 25%, at least about50%, at least about 75%, and may increase expression 2-fold, 3-fold,5-fold or more, relative to an unstimulated cell. The level ofphagocytosis in a cell thus treated may be at least about 25%, at leastabout 50%, at least about 75%, and may increase phagocytosis 2-fold,3-fold, 5-fold or more, relative to an unstimulated cell. In thepresence of an agent that blocks the interaction of CD47 with SIRPα, theincremental increase in phagocytosis for a cell treated with aneffective dose of a TLR agonist or a BTK agonist may be at least about25%, at least about 50%, at least about 75%, and may increasephagocytosis 2-fold, 3-fold, 5-fold or more, relative to a cell treatedwith a TLR agonist in the absence of CD47 blockade. In some embodimentsthe CD47 antagonist is an antibody. In some embodiments the antibody ishu5F9-G4.

For in vivo treatment, a TLR agonist or a BTK agonist may beadministered in an effective dose and for a period of time sufficient toincrease PrCr in the recipient, e.g. as determined by the phagocytosisof tumor cells by the phagocytic cells. The TLR agonist or a BTK agonistmay be co-administered or concurrently administered with an effectivedose of an agent that blocks the interaction of CD47 with SIPα. The TLRagonist or a BTK agonist may be co-administered or concurrentlyadministered with an agent that specifically targets a cancer cell, e.g.an antibody directed to a tumor selective target.

Phagocytic cells that have been treated in vitro with a TLR agonist or aBTK agonist can be administered to an individual for treatment ofcancer, where the cells are administered systemically or locally, e.g.at a tumor site. The cells may be co-administered or concurrentlyadministered with an effective dose of an agent that blocks theinteraction of CD47 with SIPα. The cells may be contacted with a tumorcell or tumor cell antigen in vitro prior to administration. The cellsmay be co-administered or concurrently administered with an agent thatspecifically targets a cancer cell, e.g. an antibody directed to a tumorselective target.

The phagocytic capability of a phagocyte, e.g. a macrophage, can bedetermined by measuring the expression of CRT on the cell surface, wherean increase in CRT corresponds to an increase in phagocytic ability. Insome embodiments, the expression of calreticulin on a macrophage cellsurface is measured, including without limitation by contacting the cellwith a CRT-specific antibody, and determining the quantity of antibodythat is bound, e.g. by flow cytometry, ELISA, immunohistochemistry, andthe like as known in the art. In some such embodiments the measuringstep is performed after treating the cells with a TLR agonist in vitro.In some embodiments, the measuring is compared to a pre-determinedlevel, or a control cell that is not treated with a TLR agonist. In someembodiments, cells that have a predetermined level of CRT areadministered to an individual for treatment of cancer, where the cellsare administered systemically or locally, e.g. at a tumor site.

In other embodiments of the invention, an inhibitor of BTK, includingwithout limitation ibrutinib, anti-BTK antibody, etc., is provided in atherapeutic dose to an individual suffering from excessive or otherwiseundesirable PrCR, including without limitation an individual sufferingfrom a myelodysplastic syndrome (MDS), autoimmune hemolytic anemia,immune thrombocytopenic purpura (ITP), autoimmune diseases includingrheumatoid arthritis, systemic lupus erythematosus, etc. The dose of BTKinhibitor is sufficient to downregulate expression of CRT on phagocyticcells, e.g. decrease by least about 25%, at least about 50%, at leastabout 75%, and may decrease expression 2-fold, 3-fold, 5-fold or more,relative to an unstimulated cell. The level of phagocytosis in a cellthus treated may be reduced by at least about 25%, at least about 50%,at least about 75%, and may decrease phagocytosis 2-fold, 3-fold, 5-foldor more, relative to an unstimulated cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A-1B. Activation of TLR signaling leads to enhanced PrCR of livingcancer cells. (FIG. 1A) Left, a schematic showing PrCR of living tumorcells by macrophages. Blockade of CD47 leads to an imbalance of “eat me”over “don't eat me” pathways, which elicits phagocytosis of tumor cells,either Fc-dependent (elicited by Fc-FcR interaction) or Fc-independent(labeled in red, representing cancer-specific “eat me” signals otherthan Fc). Right, a phagocytosis assay showing blockade of CD47 inducedphagocytosis, with SW620 cells (Control IgG-treated, anti-CD47 antibody(B6H12)-treated or CD47^(KO)) as target cells and BMDMs from RAG2^(−/−),γc^(−/−) mice. Fc-receptor blocker (FcRB) reversed phagocytosis ofB6H12-treated cells to the same level as that of CD47^(KO)) cells. **,P<0.01, t-test. (FIG. 1B) A phagocytosis assay showing a screen of TLRagonists, with SW620 cells (PBS-treated, anti-CD47 antibody(Hu5F9-G4)-treated or CD47^(KO)) as target cells and BMDMs from BALB/cmice. TLR agonists used in the screen were: Pam3CSK4 (Pam, TLR1/2), HeatKilled Listeria monocytogenes (HKLM, TLR2), Poly (I:C) HMW (Poly (I:C),TLR3), Lipopolysaccharide (LPS, TLR4), Flagellin from S. typhimurium(FLA-ST, TLR5), Pam2CGDPKHPKSF (FSL-1, TLR6/2), Imiquimod (Imi, TLR7),Class B CpG oligonucleotide (ODN 1826, TLR9). Dash lines indicate 2-foldphagocytosis of each condition (PBS-treated, anti-CD47 antibody(Hu5F9-G4)-treated or CD47^(KO)) in the control macrophage group. Errorbars represent standard deviation (A and B).

FIG. 2A-2D. Btk is the key signaling molecule regulating PrCR of cancercells; (FIG. 2A) A phagocytosis assay showing a screen with combined TLRagonists and various inhibitors targeting downstream signalingmolecules, with SW620 cells (control or CD47^(KO)) as target cells andBMDMs from RAG2^(−/−), γc^(−/−) mice. Inhibitors used in the screenwere: PD98059 (PD, MEK inhibitor), LY294002 (LY, P13K inhibitor),Ibrutinib (Ibr, Btk inhibitor), YVAD (YVAD, Caspase-1 inhibitor). **,P<0.01 (t-test; Comparison between samples in control or CD47^(KO)groups, Imi-ctrl vs other conditions). (FIG. 2B) Immunoblots showing thephosphorylation of Btk induced by TLR agonists (Poly (I:C) HMW, LPS,imiquimod). When cells were treated with TLR agonists and Ibrutinibsimultaneously, the induction of Btk phosphorylation was attenuated.Total Btk showed no change. (FIG. 2C) and (FIG. 2D) Temporal effects ofBtk activator (imiquimod) (FIG. 2C) and inhibitor (ibrutinib) (FIG. 2D)on phagocytosis, with SW620 cells^(CD47KO) as target cells and BMDMsfrom NSG mice. Error bars represent standard deviation (FIGS. 2A, 2C and2D).

FIG. 3A-3D. Btk controls cell surface exposure of CRT on macrophages toregulate PrCR of cancer cells. (FIG. 3A) The expression of CRT onmacrophages was examined by cell surface biotinylation assay.Immunoblots showed that cell surface CRT increased upon Btk activationand decreased upon Btk inhibition. Imi: imiquimod; Ibr: ibrutinib. (FIG.3B) Increased cell surface exposure of CRT on macrophages induced by TLRagonists (Poly (I:C) HMW, LPS, imiquimod), as examined by flow cytometryanalyses. Dash lines indicate normalized phagocytic indexes of eachcondition (PBS-treated, anti-CD47 antibody (Hu5F9-G4)-treated orCD47^(KO)) in the control macrophage group. (FIG. 3D) Overexpression ofCRT in J774 cells promoted phagocytosis. Expression of CRT was examinedby immunoblotting. SW620 cells (Control IgG- or anti-CD47 antibody(B6H12)-treated) were used as target cells. * P<0.05, ** P<0.01(t-test). Error bars represent standard deviation (FIGS. 3C and 3D).

FIG. 4A-4C. CRT is a key effector on macrophages in mediating PrCR ofcancer cells. (FIG. 4A) A phagocytosis assay showing the effects ofblocking CRT on macrophages or cancer cells. Left, a schematic showingthe design of the experiments. Macrophages, target cells or both werepre-treated with CRT antibody and then subjected to phagocytosis assay.Right, a phagocytosis assay showing CRT on macrophages was necessary forphagocytosis of cancer cells, with SW620 cells (control or CD47^(KO)) astarget cells and BMDMs from RAG2^(−/−), γc^(−/−) mice. (FIG. 4B)Phagocytic ability of macrophages with differential surface CRTexpression levels. Definition of CRT^(Low), CRT^(Mediumm) and CRT^(High)populations were described in FIG. 58A-B. (FIG. 4C) Normalized tumorcell phagocytosis (Y axis) were plotted against normalized cell surfaceCRT expression (Log 2; X axis) on macrophages, with SW620cells)(CD47^(KO)) as target cells and BMDMs from RAG2^(−/−), γc^(−/−) orNSG mice. ▪: BMDMs from NSG mice treated with imiquimod for 0, 1, 6, 16,24 hr; ▴ BMDMs from RAG2^(−/−), γc^(−/−) mice (CRT^(Low), CRT^(Medium),CRT^(High) and bulk populations);  BMDMs from NSG mice (CRT^(Low),CRT^(Medium), CRT^(High) and bulk populations). Error bars representstandard deviation (A and 8).

FIG. 5A-5B. TALEN-mediated CD47 knockout in SW620 cells. (FIG. 5A) and(FIG. 5B) Examination of cell surface CD47 in SW620^(WT) andSW620^(CD47KO) cells by flow cytometry analyses. Cells were stained withPhycoerythrin (PE) conjugated anti-CD47 antibody (B6H12) orPE-conjugated isotype control. Flow cytometry analyses were displayed inhistogram (FIG. 5A) or contour (FIG. 5B).

FIG. 6A-6C. Screen of TLR signaling for PRCR of tumor cells with TLRagonists. (FIG. 6A) and (FIG. 6B) Phagocytosis assays showing treatmentof macrophages with TLR agonists (Poly (I:C) HMW, LPS, imiquimod)promoted phagocytosis of tumor cells, with SW620 cells (PBS-treated,anti-CD47 antibody (Hu5F9-G4)-treated or CD47^(KO)) as target cells andBMDMs from RAG2^(−/−), γc^(−/−) mice (FIG. 6A) or NSG mice (FIG. 6B).Dash lines indicate 2-fold phagocytosis of each condition (SW620+PBS,SW620+Hu5F9-G4 or SW620^(CD47KO)+PBS) in the control macrophage group.(FIG. 6C) Representative flow cytometry plots showing TLR agonistsenhanced phagocytosis. A phagocytosis assay showing TLR agonist enhancedphagocytosis of cancer cells, with SW620 cells (PBS- orHu5F9-G4-treated) as target cells and BMDMs from NSG mice. Phagocytosiswas examined by flow cytometry analyses. Macrophages were stained withPE cy7-conjugated anti-F4/80 antibody and SW620 cells were labeled withGFP. Cells in the square of right-top corners were F4/80+GFP+ cells,representing macrophages that had phagocytosed cancer cells. Treatmentof macrophages with LPS strongly enhanced their phagocytic ability.Error bars represent standard deviation (FIGS. 6A and 6B).

FIG. 7A-7D. Phagocytosis of HL60, Raji, PC-3, MDA-MB-231 is enhanced byTLR agonists. Phagocytosis assays showing TLR agonists enhancedphagocytosis of multiple human cancer cells, with differenthematopoietic (HL60 and Raji) and solid tumor (PC3 and MDA-MB-231) cells(PBS- or Hu5F9-G4-treated) as target cells and BMDMs from NSG (HL60,Raji and PC3) or RAG2^(−/−), γc^(−/−) (MDA-MB-231) mice. HL60:Promyelocytic leukemia; Raji: Burkitt's lymphoma; PC3: Prostate cancer;MDA-MB-231: Breast cancer. Error bars represent standard deviation (FIG.7A-7D).

FIG. 8A-8B. TLR agonists improve the efficacy of CD47 blocking antibodyto inhibit the growth of tumors in vivo. (FIG. 8A) Tumor growthmonitored by bioluminescent imaging. PC3 prostate cancer cells wereengrafted in NSG mice. Mice were treated with PBS, Hu5F9-G4, orHu5F9-G4+TLR agonists (Poly (I:C) HMW+LPS) (n=5 in each group; 1 mousein the PBS control group died due to tumor progression at week 8). TLRagonists significantly enhanced the efficacy of Hu5F9-G4 to inhibittumor growth. (FIG. 8B) Analysis of tumor specimens by flow cytometry.Tumor specimens from each group in the experiment described in (FIG. 8A)were collected and dissociated to achieve single cell suspension. Thecells were analyzed by flow cytometry. Anti-CD31 and anti-Gr-1antibodies were used to exclude endothelial cells (CD31) and neutrophil(Gr-1). Macrophages were labeled with anti-F4/80 antibody. a4(GFP+F4/80−) represents tumor cells, al (GFP-F4/80+) representsmacrophages and a2 (GFP+F4/80+) represents macrophages that hadphagocytosed tumor cells. Less tumor cells were observed in Hu5F9-G4group (40%) as compared to PBS group (56%), while tumor cells werealmost cleared in Hu5F9-G4+TLR agonists group (1.06%). Hu5F9-G4 groupshowed ongoing phagocytosis (a2) (39.3%, vs 2.18% in PBS group), whileHu5F9-G4+TLR agonists group showed largely completed phagocytosis oftumor cells (with 94.7% macrophages (a1+a2), vs 46.26% in Hu5F9-G4 groupand 23.8% in PBS group). These results suggested Hu5F9-G4 enhanced theefficacy of Hu5F9-G4 in inducing PrCR of tumor cells in vivo.

FIG. 9. Expression of Btk in the hematopoietic system. A schematicshowing the expression of Btk in the hematopoietic system, generated byGene Expression Commons (5). Gene expression activity was labeled withblue (low) or red (high). Btk is expressed in all linages except for TCells and NK cells. HSC: Hematopoietic Stem Cell; MPP: MultipotentialProgenitor; GMLP: Granulo/Macrophage/Lymphoid Progenitor; pMEP/MEP:pre-/Megakaryocyte-erythroid Progenitor; CMP: Common Myeloid Progenitor;CLP: Common Lymphoid Progenitor; Plt: Platelet; Ery: Erythroid;pGMP/GMP: pre-/Granulocyte-Macrophage Progenitor; MkP:Megakaryocyte-committed Progenitor; pCFU-E: preCFU-E; Gra: Granulocyte;Mono: Monocyte; BLP: B Lymphocyte Progenitor; iNK/mNK:intermediate/mature Natural Killer Cell; BM: Bone Marrow; Spl: Spleen.

FIG. 10A-10E. Btk mediates PrCR by regulating cell surface exposure ofCRT on macrophages. (FIG. 10A) Inhibition of basal level PrCR (restingmacrophages) by Btk antagonism. A phagocytosis assay showing blockade ofBtk inhibited tumor cell phagocytosis by resting macrophages(unstimulated by TLR signaling), with SW620 cells (Control IgG-treated,anti-CD47 antibody (B6H12)-treated or CD47^(KO)) as target cells andBMDMs from RAG2^(−/−), γc^(−/−) mice. Macrophages and target cancercells were cocultured for 16 hr under indicated conditions, with orwithout Btk blocker ibrutinib. The cells were examined by flow cytometryanalyses. Remaining target cells were used to evaluate efficacy ofphagocytosis, with less remaining target cells representing strongerphagocytosis. Both F-dependent and Fc-independent phagocytosis inducedby CD47 blockade (anti-CD47 antibody or CD47^(KO)) were largely reversedby Btk antagonism. (FIG. 10B) Cell surface expression of CRT wasexamined by flow cytometry analyses. Macrophages (ctrl, imiquimod, orimiquimod+ibrutinib) were analyzed with anti-CRT antibody. Imiquimodincreased cell surface expression of CRT on macrophages and this effectwas reversed by ibrutinib. (FIG. 10C) Dose response of CRT antibody inblocking phagocytosis. Dose response curves of CRT antagonism with CRTantibody or rabbit IgG (control) in blocking phagocytosis. Phagocytosisassay was performed with SW620^(CD47KO) as target cells and BMDMs fromRAG2^(−/−), γc^(−/−) mice. (FIG. 10D) A phagocytosis assay showing CRTantibody or ibrutinib inhibited phagocytosis of cancer cells, with SW620cells (Control IgG- or anti-CD47 antibody (B6H12)-treated) as targetcells and human PBMC-derived macrophages. **, P<0.01 (t-test). (FIG.10E) Phosphorylation of CRT by Btk. Immunoblot showing CRTphosphorylation upon Btk activation. Myc-tagged CRT was expressed inJ774 cells and immunoprecipitated with anti-myc antibody after imiquimodtreatment. Phosphorylated CRT was detected with anti-phosphotyrosineantibody. Error bars represent standard deviation (FIGS. 10A, 10C and10D).

FIG. 11A-11D. Induction of cell surface CRT by Btk activation isspecific in macrophages. (FIG. 11A) Immunoblot of Btk expression inmacrophages and cancer cells. Btk was expressed in macrophages but notin solid tumor cells. 620: SW620; 231: MDA-MB-231. (FIG. 11B-11D) Cellsurface expression of CRT with or without Btk activation, as examined byflow cytometry, in macrophages (FIG. 11B), colon cancer (SW620, FIG.11C) and breast cancer (MDA-MB-231, FIG. 11D) cells. Imi, imiquimod.

FIG. 12A-120. Surface CRT expression on macrophages is correlated withtheir phagocytic abilities. (FIG. 12A) and (FIG. 12B) Macrophagesub-populations with differential cell surface CRT expression. FACSplots showing CRT expression on BMDMs from RAG2^(−/−), γc^(−/−) (FIG.12A) or NSG (FIG. 12B) mice, under control condition or imiquimodtreatment. CRT^(Low), CRT^(Medium) and CRT^(High) populations weredefined and labeled as areas a, b and c. Phagocytic ability of differentgroups of untreated macrophages (Bulk—entire population; CRT^(Low)—cellsin region a; CRT^(Medium)—cells in region b; CRT^(High)—cells in regionsc) were showed in FIG. 4B. (FIG. 12C) Temporal effects of imiquimod (0,1, 6, 16, 24 hr treatment) on cell surface expression of CRT on BMDMsfrom NSG mice, as examined by flow cytometry analysis with anti-CRTantibody. (FIG. 12D) Mean fluorescence intensity values of CRT atdifferent time points after imiquimod treatment were normalized to thevalue of 0 hr and log transformed (Log 2).

FIG. 13A-13D. Cell surface expression of CRT on M1 and M2 humanmacrophages. (A) and (8) Differentiation of M1 (FIG. 13A) and M2 (FIG.13B) macrophages was examined with specific markers (CD80 for M1 andCD163 for M2). (FIG. 13C) and (FIG. 13D) FACS plot showing cell surfaceexpression of CRT on M1 and M2 macrophages.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Calreticulin. Calreticulin is a multifunctional protein of 417 aminoacids, molecular weight 48 kDa, that binds Ca²⁺ ions, rendering itinactive. The Ca²⁺ is bound with low affinity, but high capacity, andcan be released on a signal. Calreticulin can be located in storagecompartments associated with the endoplasmic reticulum, where it bindsto misfolded proteins and prevents them from being exported to the Golgiapparatus. Calreticulin is also found in the nucleus, suggesting that itmay have a role in transcription regulation. Calreticulin binds to thesynthetic peptide KLGFFKR, which is almost identical to an amino acidsequence in the DNA-binding domain of the superfamily of nuclearreceptors. The gene symbol for calreticulin is CALR, and the humansequences may be accessed at Pubmed as follows: Protein Accession#NP_004334; Nucleotide Accession#: NM_004343.

Anti-CD47 agent. As used herein, the term “anti-CD47 agent” refers toany agent that reduces the binding of CD47 (e.g., on a target cell) toSIRPα (e.g., on a phagocytic cell). Non-limiting examples of suitableanti-CD47 reagents include SIRPα reagents, including without limitationhigh affinity SIRPα polypeptides, anti-SIRPα, antibodies, soluble CD47polypeptides, and anti-CD47 antibodies, antibody fragments, peptides,small molecules, peptidomimetics, and the like. In some embodiments, asuitable anti-CD47 agent (e.g. an anti-CD47 antibody, a SIRPα reagent,etc.) specifically binds CD47 to reduce the binding of CD47 to SIRPα. Insome embodiments, a suitable anti-CD47 agent (e.g., an anti-SIRPα,antibody, a soluble CD47 polypeptide, etc.) specifically binds SIRPα toreduce the binding of CD47 to SIRPα. A suitable anti-CD47 agent thatbinds SIRPα does not activate SIRPα (e.g., in the SIRPα-expressingphagocytic cell).

The efficacy of a suitable anti-CD47 agent can be assessed by assayingthe agent. In an exemplary assay, target cells are incubated in thepresence or absence of the candidate agent. An agent for use in themethods of the invention will up-regulate phagocytosis and subsequent Tcell activation by at least 10% (e.g., at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 120%, at least 140%, at least 160%,at least 180%, or at least 200%) compared to phagocytosis and subsequentT cell activation in the absence of the agent. Similarly, an in vitroassay for levels of tyrosine phosphorylation of SIRPα will show adecrease in phosphorylation by at least 5% (e.g., at least 10%, at least15%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or 100%) compared tophosphorylation observed in absence of the candidate agent.

In some embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) may occur (Manna and Frazier (2004) CancerResearch 64:1026-1036). Thus, in some embodiments, the anti-CD47 agentdoes not directly induce cell death of a CD47-expressing cell.

SIRPα reagent. A SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. A suitable SIRPα reagentreduces (e.g., blocks, prevents, etc.) the interaction between thenative proteins SIRPα and CD47. The SIRPα reagent will usually compriseat least the d1 domain of SIRPα. In some embodiments, a SIRPα reagent isa fusion protein, e.g., fused in frame with a second polypeptide. Insome embodiments, the second polypeptide is capable of increasing thesize of the fusion protein, e.g., so that the fusion protein will not becleared from the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPα reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules.

In some embodiments, a subject anti-CD47 agent is a “high affinity SIRPαreagent”, which includes SIRPα-derived polypeptides and analogs thereof.High affinity SIRPα reagents are described in international applicationPCT/US13/21937, which is hereby specifically incorporated by reference.High affinity SIRPα reagents are variants of the native SIRPα protein.In some embodiments, a high affinity SIRPα reagent is soluble, where thepolypeptide lacks the SIRPα transmembrane domain and comprises at leastone amino acid change relative to the wild-type SIRPα sequence, andwherein the amino acid change increases the affinity of the SIRPαpolypeptide binding to CD47, for example by decreasing the off-rate byat least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold,at least 500-fold, or more.

A high affinity SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPα reagent will usually comprise at least the d1 domainof SIRPα with modified amino acid residues to increase affinity. In someembodiments, a SIRPα variant of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPα reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules. The amino acid changes thatprovide for increased affinity are localized in the d1 domain, and thushigh affinity SIRPα reagents comprise a d1 domain of human SIRPα, withat least one amino acid change relative to the wild-type sequence withinthe d1 domain. Such a high affinity SIRPα reagent optionally comprisesadditional amino acid sequences, for example antibody Fc sequences;portions of the wild-type human SIRPα protein other than the d1 domain,including without limitation residues 150 to 374 of the native proteinor fragments thereof, usually fragments contiguous with the d1 domain;and the like. High affinity SIRPα reagents may be monomeric ormultimeric, i.e. dimer, trimer, tetramer, etc.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference). Suitable anti-CD47 antibodies include fully human, humanizedor chimeric versions of such antibodies. Humanized antibodies (e.g.,hu5F9-G4) are especially useful for in vivo applications in humans dueto their low antigenicity. Similarly caninized, felinized, etc.antibodies are especially useful for applications in dogs, cats, andother species respectively. Antibodies of interest include humanizedantibodies, or caninized, felinized, equinized, bovinized, porcinized,etc., antibodies, and variants thereof.

Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds SIRPα (i.e., an anti-SIRPα,antibody) and reduces the interaction between CD47 on one cell and SIRPαon another cell. Suitable anti-SIRPα, antibodies can bind SIRPα withoutactivating or stimulating signaling through SIRPα, because activation ofSIRPα would inhibit phagocytosis. Instead, suitable anti-SIRPα,antibodies facilitate the phagocytosis of target cells. Thus, a suitableanti-SIRPα antibody specifically binds SIRPα (withoutactivating/stimulating enough of a signaling response to inhibitphagocytosis) and blocks an interaction between SIRPα and CD47. Suitableanti-SIRPα, antibodies include fully human, humanized or chimericversions of such antibodies. Similarly caninized, felinized, etc.antibodies are especially useful for applications in dogs, cats, andother species respectively. Antibodies of interest include humanizedantibodies, or caninized, felinized, equinized, bovinized, porcinized,etc., antibodies, and variants thereof.

Soluble CD47 polypeptides. In some embodiments, a subject anti-CD47agent is a soluble CD47 polypeptide that specifically binds SIRPα andreduces the interaction between CD47 on one cell and SIRPα on anothercell. A suitable soluble CD47 polypeptide can bind SIRPα withoutactivating or stimulating signaling through SIRPα. Suitable soluble CD47polypeptides facilitate the phagocytosis of target cells. Thus, asuitable soluble CD47 polypeptide specifically binds SIRPα withoutactivating/stimulating enough of a signaling response to inhibitphagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPα are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPα andinhibit the interaction between CD47 and SIRPα without stimulatingenough SIRPα activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide (SEQ IDNO:2), such that the extracellular portion of CD47 is typically 142amino acids in length, and has the amino acid sequence set forth in SEQID NO:3. The soluble CD47 polypeptides described herein also includeCD47 extracellular domain variants that comprise an amino acid sequenceat least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% (or any percentidentity not specifically enumerated between 65% to 100%), whichvariants retain the capability to bind to SIRPα without stimulatingSIRPα signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. In anexemplary embodiment, the CD47 extracellular domain lacking the signalpeptide has the amino acid sequence set forth in SEQ ID NO:1 (124 aminoacids). As described herein, signal peptides are not exposed on the cellsurface of a secreted or transmembrane protein because either the signalpeptide is cleaved during translocation of the protein or the signalpeptide remains anchored in the outer cell membrane (such a peptide isalso called a signal anchor). The signal peptide sequence of CD47 isbelieved to be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPα without stimulating SIRPα signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to SEQ ID NO:1.

Innate Immunity. The innate immune system is a primitive cellularresponse that provides for a defense of cells against pathogen antigens.Recognition of these antigens by the innate immune system may result inan inflammatory response characterized by the production of cytokinessuch as TNF, IL-1, IL-6, and IL-8; as well as gene activation of ICAM-1and E-selectin, among others.

The broad classes of pathogens, e.g. viruses, bacteria, and fungi, mayconstitutively express a set of class-specific, mutation-resistantmolecules called pathogen-associated molecular patterns (PAMPs). Thesemicrobial molecular markers may be composed of proteins, carbohydrates,lipids, nucleic acids and/or combinations thereof, and may be locatedinternally or externally. Examples include the endotoxinlipopolysaccharide (LPS), single or double-stranded RNA, and the like.

Typically PAMP receptors (PRRs) are nonclonal, i.e. expressed on allcells of a given type, and germ-line encoded, or independent ofimmunologic memory. Once bound, PRRs tend to cluster, recruit otherextracellular and intracellular proteins to the complex, and initiatesignaling cascades that ultimately impact transcription. Further, PRRsare involved in activation of complement, coagulation, phagocytosis,inflammation, and apoptosis functions in response to pathogen detection.There are several types of PRRs including complement, glucan, mannose,scavenger, and toll-like receptors, each with specific PAMP ligands,expression patterns, signaling pathways, and anti-pathogen responses.

The Toll-like receptors are type I transmembrane (TM) PRRs that possessvarying numbers of extracellular N-terminal leucine-rich repeat (LRR)motifs, followed by a cysteine-rich region, a TIR domain, and anintracellular Toll/IL-1 R (TIR) motif. The LLR domain is important forligand binding and associated signaling and is a common feature of PRRs.The TIR domain is important in protein-protein interactions and istypically associated with innate immunity. The TIR domain also unites alarger IL-1 R/TLR superfamily that is composed of three subgroups. Thehuman TLR family is composed of at least 10 members, TLR1 through 10.Each TLR is specific in its expression patterns and PAMP sensitivities.

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) andmimetics thereof, a molecular pattern associated with viral infection.It maps to chromosome 4q35 and its sequence encodes a putative 904 aaprotein with 24 N-terminal LRRs and a calculated molecular weight of 97kDa. TLR3 is most closely related to TLR5, TLR7, and TLR8, each with 26%overall aa sequence identity. TLR3 mRNA is elevated after exposure toGram-negative bacteria and to an even greater extent in response toGram-positive bacteria.

TLR3 specifically recognizes double-stranded RNA (dsRNA) and inducesmultiple intracellular events responsible for innate antiviral immunityagainst a number of viral infections. The predicted 904-amino acid TLR3protein contains the characteristic Toll motifs: an extracellularleucine-rich repeat (LRR) domain and a cytoplasmic interleukin-1receptor-like region.

Exposure to double-stranded RNA (dsRNA) or polyinosine-polycytidylicacid (poly(I:C)), a synthetic dsRNA analog, induces the production ofinterferon α and β and by signaling through TLR3 activates NFκB. IRF3 isspecifically induced by stimulation of TLR3 or TLR4, which mediates aspecific gene program responsible for innate antiviral responses. TRIFis necessary for TLR3-dependent activation of NFKB. It serves as anadaptor protein linking RIP1 and TLR3 to mediate TLR3-induced NFKBactivation.

Toll-like receptor 4 is a protein that in humans is encoded by the TLR4gene. It detects lipopolysaccharide from Gram-negative bacteria and isthus important in the activation of the innate immune system. Thisreceptor is most abundantly expressed in placenta, and in myelomonocyticsubpopulation of the leukocytes. The human TLR4 gene may be accessed atGenbank NM_003266.3 and the protein accessed at Genbank NP_003257.1.

Activation of TLR4 leads to downstream release of inflammatorymodulators including TNF-a and Interleukin-1. Agonists include morphine,oxycodone, fentanyl, methadone, lipopolysaccharides (LPS),carbamazepine, oxcarbazepine, etc.

TLR agonist. TLR agonists activate TLRs, including without limitationTLR3, TLR4, and RIG1. Examples of TLR agonists includepathogen-associated molecular patterns (PAMPs) and mimetics thereof.These microbial molecular markers may be composed of proteins,carbohydrates, lipids, nucleic acids and/or combinations thereof, andmay be located internally or externally, as known in the art. Examplesinclude LPS, zymosan, peptidoglycans, flagellin, synthetic TLR2 agonistPam3cys, Pam3CSK4, MALP-2, Imiquimod, CpG ODN, and the like.

TLR3 agonists include double-stranded RNA; Poly(I:C), Poly(A.U), etc.,where such nucleic acids usually have a size of at least about 10 bp, atleast about 20 bp, at least about 50 bp and may have a high molecularweight of from about 1 to about 20 kb, usually not more than about 50 to100 kb. Alternative TLR3 agonists may directly bind to the protein, e.g.antibodies or small molecules that selectively bind to and activateTLR3. Other TLR3 agonists include retroviruses, e.g. a retrovirusengineered to lack the ability to integrate into the genome.

The dose of agonist that is effective in the methods of the invention isa dose that increases the expression of CRT on a phagocytic cell or cellpopulation, relative to the same population in the absence of the TLRagonist.

For example, where the TLR agonist of poly I:C or an analog thereof, aneffective dose may be at least about 10 ng/ml, at least about 50 ng/ml,at least about 100 ng/ml, at least about 250 ng/ml, at least about 500ng/ml. The dose of a TLR agonist other than poly I:C may be calculatedbased on the provision of activity equivalent to the optimized poly I:Cdose.

TLR3, 4, 7/8 and 9 agonists are of particular interest asimmunotherapeutic agents to treat cancer. Included in the group are,without limitation: 852A: Synthetic imidazoquinoline mimicking viralssRNA; VTX-2337: Small-molecule selective TLR8 agonist mimicking viralssRNA; BCG: Bacillus of Calmette-Guerin, Mycobacterium bovis; CpG ODN:CpG oligodeoxynucleotide; Imiquimod: Synthetic imidazoquinolinemimicking viral ssRNA; LPS: Lipopolysaccharide; MPL: Monophosphoryllipid A; Poly I:C: Polyriboinosinic-polyribocytidylic acid; PolyICLC:Poly I:C-poly-1-lysine; Resiquimod: Synthetic imidazoquinoline mimickingviral ssRNA.

Imiquimod is a synthetic imidazoquinoline that targets TLR7. A newerimidazoquinoline TLR7 agonist, 852A, administered parenterally asmonotherapy has shown modest clinical efficacy with diseasestabilization as a monotherapy. Resiquimod is an imidazoquinoline TLR7/8agonist in humans.

CpG are single-strand oligodeoxynucleotides (ODNs), characterized bymotifs containing cytosines and guanines. Based on their immunologiceffects, CpG ODNs are divided into three different classes: CpG-A, apotent stimulator of NK cells owing to its IFN-α-producing effect onpDCs; CpG-B, a moderate IFN-α inducer, and enhancer of antigen-specificimmune responses (upregulates costimulatory molecules on pDCs and Bcells, induces Th1 cytokine production and stimulates antigenpresentation by pDCs); and CpG-C, which combines the stimulatorycapacity of both CpG-A and CpG-B. CpG 7909 (PF-3512676, a CpG type B andTLR9 agonist) has been evaluated in several tumor types including renalcell carcinoma, glioblastoma, melanoma, cutaneous T-cell lymphoma andnon-Hodgkin's lymphoma.

Polyriboinosinic-polyribocytidylic acid (Poly I:C) is a synthetic analogof viral dsRNA that stimulates endosomal (TLR3) and/or cytosolicmelanoma differentiation-associated gene 5 (MDA5), leading to increasedproduction of type I IFNs.

Lipid A molecules that target the TLR4 complex include monophosphoryllipid A (MPL), a derivative of lipid A from Salmonella minnesota.

Bruton's tyrosine kinase (Btk) contains a PH domain that bindsphosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 binding inducesBtk to phosphorylate phospholipase C, which in turn hydrolyzes PIP2, aphosphatidylinositol, into two second messengers, inositol triphosphate(IP3) and diacylglycerol (DAG), which then go on to modulate theactivity of downstream proteins during B-cell signalling. Mutations inthe BTK gene are implicated in the primary immunodeficiency diseaseX-linked agammaglobulinemia (Bruton's agammaglobulinemia). Patients withXLA have normal pre-B cell populations in their bone marrow but thesecells fail to mature and enter the circulation. Ibrutinib (PCI-32765),is a selective Bruton's tyrosine kinase inhibitor.

Ibrutinib(1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one)is a specific inhibitor of Btk. In the methods of the present invention,it may be administered, e.g. in an oral dosage form, at a dose of fromabout 10 mg/day, about 50 mg/day, about 100 mg/day, about 250 mg/day,about 350 mg/day, about 420 mg/day, about 500 mg/day, about 600 mg/dayand not more than about 1000 mg/day. Administration may be continueduntil unacceptable toxicity or disease progression.

Phagocytic antigen presenting cell. The terms “phagocytic cells” and“phagocytes” are used interchangeably herein to refer to a cell that iscapable of phagocytosis, i.e. engulfing a large particulate mass, forexample from about 0.1 μm in diameter up to about 2 mm or about 1 mm indiameter; from about 0.5 μm in diameter in to about 1 mm in diameter,etc, particularly including up to the size of a mammalian cell, e.g. atumor cell. Phagocytosis in this context is defined by the engulfment ofcells, pathogens, and various particles by surrounding it with theeffector cell membrane.

There are several categories of phagocytes: macrophages; mononuclearcells (histiocytes and monocytes); polymorphonuclear leukocytes;(neutrophils) and dendritic cells. Macrophages are of particularinterest. Phagocytosis-associated cell responses includeimmunomodulatory responses like the generation and release ofpro-inflammatory and anti-inflammatory mediators, and also cellresponses of destructive nature such as the respiratory burst, and therelease of toxic and microbicidal molecules by degranulation.Professional phagocytes are capable of recognizing a wide variety ofphagocytic targets, and of ingesting them at a higher rate thannon-phagocytic cells.

Neutrophils and macrophages are representative of fully differentiatedphagocytes. While neutrophils leaving the bone marrow are fullydifferentiated, macrophages differentiate from circulating monocytes inextra-vascular tissues. Monocytes display a lower phagocytic response,compared to neutrophils and macrophages, and must respond to activationand differentiation signals in order to achieve optimal phagocyticcapacity. The process of monocyte-to-macrophage differentiation has beenwell characterized, and can be performed in vitro or in vivo.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy). For some purposes in the present invention, an effective doseof an anti-CD47 agent is the dose that increases phagocytosis by atleast about 10%, at least about 20%, at least about 50%, at least about75%, at least about 100%, up to 2-fold, 3-fold or more.

For purposes of this invention, a therapeutically effective dose of ananti-CD47 agent is an amount that is sufficient to palliate, ameliorate,stabilize, reverse, prevent, slow or delay the progression of thedisease state (e.g., cancer or chronic infection) by increasingmacrophage mediated killing of a target cell. Thus, a therapeuticallyeffective dose of an anti-CD47 agent can decrease the target cellpopulation through an in vivo immune response by at least about 10%, atleast about 20%, at least about 50%, at least about 75%, at least about90% or more, relative to the effect in the absence of administering aloaded population of phAPC.

Myelodysplastic syndromes. The myelodysplastic syndromes (also known asMDS or myelodysplasia) are hematological (i.e., blood-related) medicalconditions with ineffective development (or “dysplasia”) of blood cells.Patients with MDS can develop severe anemia and require bloodtransfusions. In some cases, the disease worsens and the patientdevelops cytopenias (low blood counts) caused by progressive bone marrowfailure. The outlook in MDS depends on the type and severity.

Included as types of MDS that can be treated by the methods of theinvention is refractory anemia; refractory anemia with ringsideroblasts; refractory anemia with excess blasts; refractory cytopeniawith multilineage dysplasia; refractory cytopenia with unilineagedysplasia; unclassifiable myelodysplastic syndrome; myelodysplasticsyndrome associated with an isolated del(5q) chromosome abnormality;chronic myelomonocytic leukemia (CMML).

Autoimmune hemolytic anemia (AIHA) is defined as an increaseddestruction of erythrocytes due to the presence of anti-erythrocyteautoantibodies (AEA) and can be classified as either autoimmune,alloimmune, or drug-induced depending on the type of antigen giving riseto the immune response. General hemolytic anemia is estimated to occurin about 4 cases per 1000 per year, but for AIHA the annual incidence isestimated to about 1-3 cases per 100,000 per year. AIHA can appeareither as a primary disease or, in about 20-80% of the cases, secondaryto other autoimmune diseases, lymphoid malignancies, infections,immunodeficiencies, or tumors, where lymphoid malignancies are the mostcommon reasons for secondary AIHA. AEA are classified as cold or warmautoantibodies, as they react optimally at temperatures below 30° C. orat 35° C. to 40° C. respectively. Warm AEA are mostly IgG but sometimesIgA and/or IgM are also present, and are responsible for about 50-70% ofAIHA cases. The binding of warm IgG AEA to erythrocytes does not itselfdamage the erythrocytes, since erythrocyte bound IgG, in contrast tosurface bound IgM, is a poor activator of the classical complementpathway. Instead, surface bound IgG is usually recognized by Fcγreceptors of cells of the monocyte-macrophage phagocytic system,preferentially in the spleen and liver, resulting in uptake anddestruction of IgG-opsonized erythrocytes. However, macrophage-mediatedelimination of erythrocytes in AIHA is likely to be mediated bysynergistic activity of macrophage Fcγ and complement receptors(recognizing complement factors C3b and C3b_(i)), since erythrocytesopsonized with very low levels of IgG are not eliminated in vivo in theabsence of complement. Furthermore, low levels of complementopsonization does not result in erythrocyte phagocytosis in the absenceof IgG, whereas low levels of both complement and IgG-opsonization caninduce efficient erythrocyte phagocytosis both in vivo and in vitro.

Immune thrombocytopenic purpura (ITP) is an autoimmune diseasecharacterised by low platelet counts due to antibody-mediateddestruction of platelets by macrophages. ITP is classified as acute orchronic, where acute ITP has a rapid onset with typical petechiae andbruises, is often preceded by an infectious illness, mainly affectsyoung children, and normally resolves spontaneously within six months.Chronic ITP often has an adult onset that is more insidious than theacute form and is about two to three times as common among women asamong men.

A positive anti-platelet autoantibody test is found in about 70-80% ofadults with ITP and in children with chronic ITP. Plateletautoantibodies are of the IgG type and are mostly directed to plateletmembrane glycoproteins, including GPIIb/IIIa, GPIb-IX, and GPIa-IIa.Platelets coated with IgG autoantibodies undergo accelerated clearancethrough Fcγ receptor-mediated phagocytosis by macrophages, preferably inthe spleen and liver. Most patients have antibodies directed to severaldifferent platelet surface proteins. Adults with diagnosed ITP areconventionally initially treated with corticosteroids. Intravenousgammaglobulin (IVIG) is another common approach in treatment of ITP,particularly for treatment of internal bleedings. IVIG has well knownanti-inflammatory effects, generally attributed to the immunoglobulin G(IgG) Fc domain, which is thought to block pro-phagocytic Fc receptorson macrophages.

The term “antibody” or “antibody moiety” is intended to include anypolypeptide chain-containing molecular structure with a specific shapethat fits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. Antibodies utilized in the present inventionmay be polyclonal antibodies, although monoclonal antibodies arepreferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

The phrase “bispecific antibody” refers to a synthetic or recombinantantibody that recognizes more than one protein. Examples includebispecific antibodies 2B1, 520C9xH22, mDX-H210, and MDX447. Bispecificantibodies directed against a combination of epitopes, will allow forthe targeting and/or depletion of cellular populations expressing thecombination of epitopes.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Freund's, Freund'scomplete, oil-in-water emulsions, etc.) When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate. Commonly utilizedconjugate proteins that are commercially available for such use includebovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In orderto raise antibodies to particular epitopes, peptides derived from thefull sequence may be utilized. Alternatively, in order to generateantibodies to relatively short peptide portions of the protein target, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as ovalbumin, BSA or KLH.

Alternatively, for monoclonal antibodies, hybridomas may be formed byisolating the stimulated immune cells, such as those from the spleen ofthe inoculated animal. These cells are then fused to immortalized cells,such as myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. In addition, the antibodies orantigen binding fragments may be produced by genetic engineering.Humanized, chimeric, or xenogeneic human antibodies, which produce lessof an immune response when administered to humans, are preferred for usein the present invention.

Antibodies that have a reduced propensity to induce a violent ordetrimental immune response in humans (such as anaphylactic shock), andwhich also exhibit a reduced propensity for priming an immune responsewhich would prevent repeated dosage with the antibody therapeutic orimaging agent are preferred for use in the invention. These antibodiesare preferred for all administrative routes. Thus, humanized, chimeric,or xenogenic human antibodies, which produce less of an immune responsewhen administered to humans, are preferred for use in the presentinvention.

Chimeric antibodies may be made by recombinant means by combining themurine variable light and heavy chain regions (VK and VH), obtained froma murine (or other animal-derived) hybridoma clone, with the humanconstant light and heavy chain regions, in order to produce an antibodywith predominantly human domains. The production of such chimericantibodies is well known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 5,624,659, incorporatedfully herein by reference). Humanized antibodies are engineered tocontain even more human-like immunoglobulin domains, and incorporateonly the complementarity-determining regions of the animal-derivedantibody. This is accomplished by carefully examining the sequence ofthe hyper-variable loops of the variable regions of the monoclonalantibody, and fitting them to the structure of the human antibodychains. Although facially complex, the process is straightforward inpractice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully hereinby reference. Alternatively, single chain antibodies (Fv, as describedbelow) can be produced from phage libraries containing human variableregions. See U.S. Pat. No. 6,174,708, incorporated fully herein byreference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ficin, pepsin, papain, or otherprotease cleavage. “Fragment” or minimal immunoglobulins may be designedutilizing recombinant immunoglobulin techniques. For instance “Fv”immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

Fv fragments are heterodimers of the variable heavy chain domain (V_(H))and the variable light chain domain (V_(L)). The heterodimers of heavyand light chain domains that occur in whole IgG, for example, areconnected by a disulfide bond. Recombinant Fvs in which V_(H) and V_(L)are connected by a peptide linker are typically stable, see, forexample, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879-5883 (1988)and Bird et al., Science 242:423-426 (1988), both fully incorporatedherein, by reference. These are single chain Fvs which have been foundto retain specificity and affinity and have been shown to be useful forimaging tumors and to make recombinant immunotoxins for tumor therapy.Any of these minimal antibodies may be utilized in the presentinvention, and those which are humanized to avoid HAMA reactions arepreferred for use in embodiments of the invention.

In addition, derivatized immunoglobulins with added chemical linkers,detectable moieties, e.g. fluorescent dyes, enzymes, radioisotopes,substrates, chemiluminescent moieties, or specific binding moieties,e.g. streptavidin, avidin, biotin, etc. may be utilized in the methodsand compositions of the present invention. For convenience, the term“antibody” or “antibody moiety” will be used throughout to generallyrefer to molecules which specifically bind to an epitope of the targetedprotein, although the term will encompass all immunoglobulins,derivatives, fragments, recombinant or engineered immunoglobulins, andmodified immunoglobulins, as described above.

Candidate binding agents can be tested for activity by any suitablestandard means. As a first screen, the antibodies may be tested forbinding against the target antigen utilized to produce them. As a secondscreen, candidate agents may be tested for binding to an appropriatecell, e.g. cancer cell, hematopoietic cell, etc. For these screens, thecandidate antibody may be labeled for detection (e.g., with fluoresceinor another fluorescent moiety, or with an enzyme such as horseradishperoxidase). After selective binding to the target is established, thecandidate agent may be tested for appropriate activity (i.e., theability to decrease tumor cell growth and/or to aid in visualizing tumorcells) in an in vivo model.

By “manipulating phagocytosis” is meant an up-regulation or adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention. Thus in the context of decreasingphagocytosis of circulating hematopoietic cells, particularly in atransplantation context, manipulating phagocytosis means adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. Cells of interestfor detection, analysis, or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. Cancers of virtually every tissue are known. Thephrase “cancer burden” refers to the quantum of cancer cells or cancervolume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g. clone of a cancercell. Many types of cancers are known to those of skill in the art,including solid tumors such as carcinomas, sarcomas, glioblastomas,melanomas, lymphomas, myelomas, etc., and circulating cancers such asleukemias. Examples of cancer include but are not limited to, ovariancancer, breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, headand neck cancer, and brain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,used interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

A “host cell”, as used herein, refers to a microorganism or a eukaryoticcell or cell line cultured as a unicellular entity which can be, or hasbeen, used as a recipient for a recombinant vector or other transferpolynucleotides, and include the progeny of the original cell which hasbeen transfected. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

“Therapeutic target” refers to a gene or gene product that, uponmodulation of its activity (e.g., by modulation of expression,biological activity, and the like), can provide for modulation of thecancerous phenotype. As used throughout, “modulation” is meant to referto an increase or a decrease in the indicated phenomenon (e.g.,modulation of a biological activity refers to an increase in abiological activity or a decrease in a biological activity).

“Reducing growth of cancer cells” includes, but is not limited to,reducing proliferation of cancer cells, and reducing the incidence of anon-cancerous cell becoming a cancerous cell. Whether a reduction incancer cell growth has been achieved can be readily determined using anyknown assay, including, but not limited to, [³H]-thymidineincorporation; counting cell number over a period of time; detectingand/or measuring a marker associated with the cancer, etc.

Whether a substance, or a specific amount of the substance, is effectivein treating cancer can be assessed using any of a variety of knowndiagnostic assays for cancer, including, but not limited to biopsy,contrast radiographic studies, CAT scan, and detection of a tumor markerassociated with cancer in the blood of the individual. The substance canbe administered systemically or locally, usually systemically.

In certain embodiments, a bi-specific antibody may be used. For examplea bi-specific antibody in which one antigen binding domain is directedagainst CTR and the other antigen binding domain is directed against acancer cell marker, such as CD47, EGFR; HER2; CD96, CD97, CD99, PTHR2,HAVCR2 etc.

For administration, the active agents will be mixed, prior toadministration, with a non-toxic, pharmaceutically acceptable carriersubstance. Usually, this will be an aqueous solution, such as normalsaline or phosphate-buffered saline (PBS), Ringers solution,lactate-Ringers solution, or any isotonic physiologically acceptablesolution for administration by the chosen means. Preferably, thesolution is sterile and pyrogen-free, and is manufactured and packagedunder current Good Manufacturing Processes (GMPs), as approved by theFDA. The clinician of ordinary skill is familiar with appropriate rangesfor pH, tonicity, and additives or preservatives when formulatingpharmaceutical compositions for administration by intravascularinjection, intrathecal injection, injection into the cerebro-spinalfluid, direct injection into the tumor, or by other routes. In additionto additives for adjusting pH or tonicity, the antibody-therapeutics andantibody-imaging agents may be stabilized against aggregation andpolymerization with amino acids and non-ionic detergents, polysorbate,and polyethylene glycol. Optionally, additional stabilizers may includevarious physiologically-acceptable carbohydrates and salts. Also,polyvinylpyrrolidone may be added in addition to the amino acid.Suitable therapeutic immunoglobulin solutions which are stabilized forstorage and administration to humans are described in U.S. Pat. No.5,945,098, incorporated fully herein by reference. Other agents, such ashuman serum albumin (HSA), may be added to the therapeutic or imagingcomposition to stabilize the antibody conjugates.

The compositions of the invention may be administered using anymedically appropriate procedure, e.g., intravascular (intravenous,intraarterial, intracapillary) administration, injection into thecerebrospinal fluid, intracavity, subcutaneously, or direct injection inthe tumor. For the imaging compositions of the invention, administrationvia intravascular injection is preferred for pre-operative visualizationof the tumor.

The effective amount of the active agents to be given to a particularpatient will depend on a variety of factors, several of which will bedifferent from patient to patient. A competent clinician will be able todetermine an effective amount to administer to a patient to retard thegrowth and promote the death of tumor cells, or an effective amount ofan imaging composition to administer to a patient to treatmentmyelodysplastic syndrome. Dosage will depend on the treatment of thetumor, route of administration, the nature of the therapeutics,sensitivity of the tumor to the therapeutics, etc. Utilizing LD₅₀ animaldata, and other information available for the agents, a clinician candetermine the maximum safe dose for an individual, depending on theroute of administration. For instance, an intravenously administereddose may be more than an intrathecally administered dose, given thegreater body of fluid into which the therapeutic composition is beingadministered. Similarly, compositions which are rapidly cleared from thebody may be administered at higher doses, or in repeated doses, in orderto maintain a therapeutic concentration. Imaging moieties are typicallyless toxic than cytotoxic moieties and may be administered in higherdoses in some embodiments. Utilizing ordinary skill, the competentclinician will be able to optimize the dosage of a particulartherapeutic composition in the course of routine clinical trials.

Typically the dosage will be 0.001 to 100 milligrams per kilogramsubject body weight. The agents can be administered to the subject in aseries of more than one administration. For therapeutic compositions,regular periodic administration (e.g., every 2-3 days) will sometimes berequired, or may be desirable to reduce toxicity. For therapeuticcompositions which will be utilized in repeated-dose regimens, agentswhich do not provoke HAMA or other immune responses are preferred.

Methods of Use

Methods are provided for the treatment of cancer. An activator of TLRsignaling or a BTK agonist is provided in combination with CD47blockade, where the removal of cancer cells is increased relative to thecell removal in the presence of either agent as a monotherapy. In someembodiments, a population of cells comprising macrophages is contactedin vitro or in vivo with a dose of a TLR agonist or a BTK agonist thatis effective in increasing CRT on the cell surface of the macrophage byat least about 25%, at least about 50%, at least about 75%, and mayincrease expression 2-fold, 3-fold, 5-fold or more, relative to anunstimulated cell. The level of phagocytosis in a cell thus treated maybe at least about 25%, at least about 50%, at least about 75%, and mayincrease phagocytosis 2-fold, 3-fold, 5-fold or more, relative to anunstimulated cell. In the presence of an agent that blocks theinteraction of CD47 with SIRPα, the incremental increase in phagocytosisfor a cell treated with an effective dose of a TLR agonist or a BTKagonist may be at least about 25%, at least about 50%, at least about75%, and may increase phagocytosis 2-fold, 3-fold, 5-fold or more,relative to a cell treated with a TLR agonist in the absence of CD47blockade.

For in vivo treatment, a TLR agonist or a BTK agonist can beadministered in an effective dose and for a period of time sufficient toincrease PrCr in the recipient, e.g. as determined by the phagocytosisof tumor cells by the phagocytic cells. The TLR agonist or a BTK agonistmay be co-administered or concurrently administered with an effectivedose of an agent that blocks the interaction of CD47 with SIPα. The TLRagonist or a BTK agonist may be co-administered or concurrentlyadministered with an agent that specifically targets a cancer cell, e.g.an antibody directed to a tumor selective target.

Phagocytic cells that have been treated in vitro with a TLR agonist or aBTK agonist can be administered to an individual for treatment ofcancer, where the cells are administered systemically or locally, e.g.at a tumor site. The cells may be co-administered or concurrentlyadministered with an effective dose of an agent that blocks theinteraction of CD47 with SIPα. The cells may be contacted with a tumorcell or tumor cell antigen in vitro prior to administration. The cellsmay be co-administered or concurrently administered with an agent thatspecifically targets a cancer cell, e.g. an antibody directed to a tumorselective target.

The phagocytic capability of a phagocyte, e.g. a macrophage, can bedetermined by measuring the expression of CRT on the cell surface, wherean increase in CRT corresponds to an increase in phagocytic ability. Insome embodiments, the expression of calreticulin on a macrophage cellsurface is measured, including without limitation by contacting the cellwith a CRT-specific antibody, and determining the quantity of antibodythat is bound, e.g. by flow cytometry, ELISA, immunohistochemistry, andthe like as known in the art. In some such embodiments the measuringstep is performed after treating the cells with a TLR agonist in vitro.In some embodiments, the measuring is compared to a pre-determinedlevel, or a control cell that is not treated with a TLR agonist. In someembodiments, cells that have a predetermined level of CRT areadministered to an individual for treatment of cancer, where the cellsare administered systemically or locally, e.g. at a tumor site.

In other embodiments of the invention, an inhibitor of BTK, includingwithout limitation ibrutinib, is provided in a therapeutic dose to anindividual suffering from excessive or otherwise undesirable PrCR,including without limitation an individual suffering frommyelodysplastic syndrome (MDS), autoimmune hemolytic anemia (AIHA),immune thrombocytopenic purpura (ITP), etc. The dose of BTK inhibitor issufficient to downregulate expression of CRT on phagocytic cells, e.g.decrease by least about 25%, at least about 50%, at least about 75%, andmay decrease expression 2-fold, 3-fold, 5-fold or more, relative to anunstimulated cell. The level of phagocytosis in a cell thus treated maybe reduced by at least about 25%, at least about 50%, at least about75%, and may decrease phagocytosis 2-fold, 3-fold, 5-fold or more,relative to an unstimulated cell.

Example 1

Macrophages Eat Cancer Cells Using their Own Calreticulin as a Guide:Roles of TLR and BTK

Macrophage-mediated programmed cell removal (PrCR) is an importantmechanism of eliminating diseased and damaged cells prior to programmedcell death. The induction of PrCR by “eat me” signals on tumor cells iscountered by “don't eat me” signals such as CD47, which binds macrophagesignal-regulatory protein α (SIRPα) to inhibit phagocytosis. Blockade ofCD47 on tumor cells leads to phagocytosis by macrophages. Here wedemonstrate that the activation of toll-like receptor (TLR) signalingpathways in macrophages synergizes with blocking CD47 on tumor cells toenhance PrCR. Bruton's tyrosine kinase (BTK) mediates TLR signaling inmacrophages. Calreticulin, previously shown to be an “eat me” signal oncancer cells, is activated in macrophages for secretion and cell surfaceexposure by TLR and Btk, to target cancer cells for phagocytosis, evenif the cancer cells do not themselves express calreticulin.

Programmed cell removal (PrCR) is a process of macrophage-mediatedimmunosurveillance by which target cells are recognized andphagocytosed. PrCR was previously known as a key step concurrent withprogrammed cell death for the clearance of apoptotic cells, but whenapoptosis is blocked, PrCR of neutrophils that are living (due toenforced expression of bcl2) occurs precisely at the same time that PrCRremoves dying wild-type neutrophils. Recently a role for PrCR ineliminating living tumor cells has been revealed. Several studies haveindicated a crucial function of CD47 as an anti-phagocytic “don't eatme” signal dominating over PrCR. During cancer development, tumor cellsupregulate CD47, which protects them from PrCR. Blockade of theinteraction between CD47 on target cells and its receptor,signal-regulatory protein α (SIRPα), on macrophages elicits efficientPrCR of cancer cells, but not most normal cells in vitro and in vivo(FIG. 1A). When CD47 is blocked, cancer cells, but not normal cells, arephagocytosed because pro-phagocytic “eat me” signals such ascalreticulin (CRT) are commonly expressed on many leukemias, lymphomas,and solid tumors (FIG. 1A).

Calreticulin is normally an endoplasmic reticulum (ER) proteinpossessing ER retention KDEL sequences, but can be released to the cellsurface in many instances of cell damage by cytotoxic drugs orinflammation and is recognized by macrophage LRP1/CD91 duringphagocytosis of apoptotic cells. Bruton's tyrosine kinase (Btk) is amember of the Tec nonreceptor protein tyrosine kinase family, whichplays a crucial role in the regulation of the innate immune response. Adefect of Btk leads to immunodeficiencies including X-linked hypo- oragammaglobulinemia, presumably due to the blockade of B celldevelopment, and perhaps related to inefficient clearance of defectiveB-lineage cells as well. So far, however, little is known concerning themolecular mechanisms by which macrophages recognize and phagocytoseliving cancer cells. We show here that macrophages express calreticulin,and that Toll-like receptor (TLR) signaling through Btk results in itstrafficking to the cell surface, where it can be used to mediate PrCR ofappropriate tumor cells.

We performed phagocytosis assays by co-culturing mouse bonemarrow-derived macrophages (BMDMs) and target human cancer cells toexamine the efficacy of PrCR under different conditions. To inducephagocytosis, we blocked CD47 on a human colon cancer cell line (SW620)by either treating tumor cells with CD47 blocking antibodies or directlyknocking it out. Phagocytosis was significantly increased by knockingout the self-protective signal CD47 (SW620^(CD74KO); FIG. 5, A-B),resulting from an imbalance of “eat me” over “don't eat me” pathways(FIG. 1A). Treatment of SW620^(WT) cells with anti-CD47 antibodyelicited stronger phagocytosis which was reversed by Fc-receptorblockers to the same level as that of SW620^(CD74KO) cells, suggestingthat anti-CD47 antibody induced phagocytosis of SW620 cancer cells byboth blockade of CD47− SIRPα interactions (Fc-independent) and withFc-dependent mechanisms (FIG. 1A).

To understand the molecular mechanisms of PrCR, we performed screeningexperiments to identify signaling pathways that regulate phagocyticability of macrophages. TLR signaling plays a crucial role in the innateimmune response against pathogens, and TLR agonists are listed asimmunotherapeutic agents with anti-cancer potential. However, the roleof TLR signaling in PrCR of living cancer cells remains unexplored.Thus, we pretreated BMDMs with various TLR agonists and then assayedtheir phagocytotic ability against cancer cells. We found that theactivation of multiple TLRs significantly enhanced phagocytosis ofcancer cells (FIG. 1B). We next focused on the TLR agonists that weremost effective at enhancing phagocytosis, assessing their effects on awider range of macrophages and tumor cells. We showed that treatment ofmacrophages with TLR3, 4 and 7 agonists (i.e.,polyinosinic-polycytidylic acid-high molecular weight (Poly (I:C) HMW),lipopolysaccharide (LPS), and imiquimod) dramatically enhanced theirphagocytosis of multiple hematopoietic and solid tumor cells (FIGS. 6A-Cand 7 A-D). Subsequent assessment in mice lacking T-, B- and NK cells,showed that these TLR agonists significantly improved the efficacy ofCD47 blocking antibody to block tumor growth in vivo (FIG. 8, A-B).

To further understand the mechanism by which the activation of TLRsignaling in macrophages promoted tumor cell phagocytosis, we treatedmacrophages by combining TLR agonists with various inhibitors targetingkey molecules that positively (MAPK, Btk) or negatively (PI3K,Caspase-1) regulate TLR signaling. Blockers of MAPK, PI3K, and Caspase1showed no effect on phagocytosis of cancer cells. In contrast,ibrutinib—a specific blocker of Bruton's tyrosine kinase (Btk), atyrosine kinase expressed in the hematopoietic system (FIG. 9),significantly attenuated phagocytosis induced by TLR agonists (FIG. 2A).Treatment of macrophages with Poly (I:C) HMW, LPS, or imiquimodstimulated Btk to be phosphorylated, and this effect was counteracted byibrutinib, resulting in basal Btk phosphorylation (FIG. 2B). Notably,basal level phagocytosis of cancer cells was regulated by the Btkpathway, and ibrutinib showed an inhibitory effect on both Fc-dependentand -independent phagocytosis (FIG. 10A). In sum, Btk is a crucialeffector through which TLRs mediate tumor cell phagocytosis.Interestingly, stimulation and inhibition of Btk showed differentialtemporal effects on phagocytosis. Maximal phagocytic ability ofmacrophages was achieved with 16 hr of Btk-activation (FIG. 2C); Incontrast, blockade of Btk showed a prompt effect and reached the maximalinhibition within 1 hr (FIG. 2D).

Upon activation, Btk phosphorylates transcription factors such as TFII-Iand STAT5A in the nucleus and PLCγ2 at the plasma membrane. Recentstudies identified CRT as a substrate phosphorylated by Btk when TLR7was activated in the recognition of apoptotic cells. Phosphorylation ofCRT by Btk in macrophages was important for CRT trafficking to the cellsurface to function as a bridging molecule in the CRT/CD91/C1q complex,which initiates phagocytosis of apoptotic cells. To investigate whetherCRT is the critical downstream effector of TLR-Btk pathway to mediatePrCR of tumor cells, we then examined the expression and function of CRTin macrophages. We found that CRT was expressed on the surface ofmacrophages, and its cell surface exposure was regulated by theactivation status of Btk (FIG. 3, A-B, FIG. S6B). CRT antibodysignificantly inhibited phagocytosis of SW620 cells by mouse BMDMs orhuman peripheral blood mononuclear cells (PBMC)-derived macrophages(FIG. 3C and FIG. 10C-D), while overexpression of CRT in a mousemonocyte/macrophage cell line J774 led to enhanced phagocytosis (FIG.3D). In addition, we confirmed phosphorylation of CRT upon Btkactivation, which reached the maximal level after 30 min of imiquimodtreatment of macrophages (FIG. 10E). These results suggest that CRT isan essential component regulated by the TLR-Btk pathway to mediatephagocytosis of living cancer cells.

We further dissected the role of CRT in mediating PrCR of cancer cells.Previous studies demonstrated cell surface expression of CRT onapoptotic cells and multiple viable human cancer cells (FIG. 11, B-D).Thus we examined whether CRT played a critical role in mediating cancercell phagocytosis on both macrophages and target tumor cells (FIG. 4A).Interestingly, blockade of CRT on macrophages diminished phagocytosisbut blocking CRT on cancer cells showed no effect, suggesting a specificrole of CRT on macrophages to mediate phagocytosis (FIG. 4A).Importantly, cell surface expression of CRT was enhanced by TLR agonistsin macrophages but not target cancer cells, which lack Btk (FIG. 11,A-D), indicating distinct mechanisms regulating CRT exposure. Next, weexamined macrophage sub-populations with different levels of cellsurface CRT and found that macrophages with a higher surface CRT showeda stronger phagocytic ability (FIG. 4B and FIG. 12A-B). Quantitativeanalysis of a panel of macrophages, including sub-populations withdifferential surface CRT expression and macrophages at different timepoints after imiquimod treatment, revealed a significant correlationbetween CRT expression on macrophages and tumor cell phagocytosis (FIG.4C and FIG. 12C-D). Additionally, M1 and M2 human macrophages derivedfrom the peripheral blood both expressed CRT on the surface, and M1subset expressed a somewhat higher level of CRT (FIG. 13). Takentogether, these findings indicate that CRT is a key effector formacrophage-mediated surveillance of tumor cells and enhanced PrCR ofcancer cells can be achieved by upregulating CRT on macrophages.

Recent progress in cancer immunology has highlighted the ability ofcancer cells to evade immunosurveillance as one of the essentialhallmarks of cancer. While lymphocytes (T, B, and NK cells) have beenthought to mediate the bulk of anti-cancer immunosurveillance, we havedemonstrated that blockade of CD47 on tumor cells leads to in vivoimmune recognition, macrophage phagocytosis of tumor cells, and tumorelimination in mice deficient in lymphocytes, indicating that phagocytesare crucial to surveillance against cancer cells. Phagocytosis of tumorcells mediated by anti-CD47 blockade can result in cross-presentation oftumor antigens to CD8 T cells, so that CD47 blockade can result in bothinnate immune system macrophage surveillance and stimulation of adaptiveimmune system T cell cytotoxicity.

Here we show that cell surface CRT on macrophages, which is controlledby the TLR-Btk pathway in causing phosphorylation of endoplasmicreticulum CRT, its cleavage from the ER retention signals withsubsequent secretion, where it is able to bind to macrophage CD91. Weshow that this mechanism of secretion is important for mediating PrCR oflive cancer cells, in addition to the demonstration of this mechanism toremove apoptotic cells. CRT on macrophages may function in detectingtarget cells through trans-interaction with as yet unidentified specificreceptors on target cancer cells; thus blockade of surface CRT inhibitsPrCR. Moreover, CD47 mutant mice do not phagocytose self red cells orhematopoietic stem cells (HSC), but these cells are rapidly phagocytosedwhen transferred to wild type congenic normal or irradiated mice, eventhough neither cell type expresses CRT in microarrays, indicating other“eat me” signals could be used, or that CRT can decorate target cellsthat do not express calreticulin genes.

We show that multiple types of TLR agonists are able to stimulatemacrophages and enhance PrCR of solid tumor cells, consistent withreports that TLR4 agonist LPS and IFN-γ receptors were necessary foractivating macrophages to phagocytose acute myeloid leukemia cells afterdisrupting CD47− SIRPα interaction. It is possible from these studiesthat TLR signaling can synergize with anti-CD47 blockade to enhancetumor cell phagocytosis, but the potential for TLR signaling of normalcells, creating them also as targets for phagocytosis, must be tested inseveral systems before one can judge the clinical potential of suchsynergy. Further investigation of the interaction between macrophagesand target cancer cells should advance our understanding of theprinciples of cancer cell immune evasion.

Materials and Methods

Mice. BALB/c, RAG2^(−/−) γc^(−/−) BALB/c and NOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG) mice were bred in a pathogen-free facility inthe Institute for Stem Cell Biology and Regenerative Medicine atStanford University. All animal procedures were approved by theAdministrative Panel on Laboratory Animal Care at Stanford University.

Cell culture. Human cancer derived cell lines SW620 (colon cancer), HL60(leukemia), Raji (lymphoma), MDA-MB-231 (breast cancer), PC3 (prostatecancer) and murine macrophage/monocyte cell line J774 were obtained fromATCC and routinely cultured in DMEM medium supplemented with 10% fetalbovine serum (SW620, MDA-MB-231, J774), IMDM medium supplemented with20% fetal bovine serum (HL60), F-12K medium supplemented with 10% fetalbovine serum (PC-3), or RPMI-1640 medium supplemented with 10% fetalbovine serum (Raji). Tumor cells were transduced with lenti viruseswhich were generated with a pCDH-CMV-MCS-EF1 lenti viral vectorexpressing a luciferase-eGFP fusion protein and sorted by flow cytometrywith BD FACSAria II cell sorters for GFP+ cells, as describedpreviously.

CD47 knockout with TALEN. TALENs were designed and assembled asdescribed. The genomic locus of human CD47 (NC_000003.12) was scannedfor putative TALEN binding pairs. Exon 2 was ultimately selected fortargeting and the TALEN pairs TGTCGTCATTCCATGCTTTG andTATACTTCAGTAGTGTTTTG were respectively cloned into the pTALEN backbone.SW620 cells were transfected with the CD47-TALEN constructs usinglipofectamine 2000. Three days after transfection, cells were stainedwith anti-CD47 or Isotype antibodies. CD47− cells were sorted by flowcytometry with BD FACSAria II cell sorters.

Flow cytometry Analysis. Flow cytometry analyses were performed using aBD LSRFortessa. For staining, 2.5×10⁵-10⁶ cells were incubated withindicated antibodies (1:50-1:200) in FACS buffer (PBS with 2% fetalbovine serum) on ice for 30 minutes. Cells were then washed with FACSbuffer and subjected to FACS analyses. For staining of macrophages,cells were first treated with Fc receptor blockers or a highconcentration of isotype IgG control (5-10 times of indicatedantibodies) to block non-specific binding of antibodies caused by theinteraction of Fc domain and Fc receptors on macrophages.

Preparation of macrophages. Human peripheral blood-derived macrophageswere generated as previously described. Monocytes were enriched fromhuman peripheral blood and differentiated to macrophages by culture inIMDM supplanted with 10% human serum for 7-10 days. To generate M1 humanmacrophages, monocytes were treated with recombinant human GM-CSF (5ng/mL) in RPMI 1640 medium supplemented with 5% FBS and 1% glutamax overall 7 days. M1 polarization was achieved by further treatment on day 5by IFN-γ (20 ng/mL) stimulation for 1 hr, followed by LPS (100 ng/mL)treatment on days 6-7. To generate M2 human macrophages, monocytes weretreated with recombinant human M-CSF (25 ng/mL) in RPMI 1640 mediumsupplemented with 5% FBS and 1% glutamax over all 7 days.M2-polarization was achieved by further treatment on day 5 and 6 withIL-4 (20 ng/mL) and IL-13 (20 ng/mL). Differentiation of M1- and M2macrophages were evaluated by the expression of specific surface makersCD80 (M1) and CD163 (M2).

Anti-CD47 (BD Biosciences), anti-calreticulin (Enzo Life Sciences, Abcamand MBL International), anti-F4/80 antibodies (Biolegend), anti-CD31antibody (BD Biosciences), anti-Gr-1 antibody (Biolegend) were used forFACS analyses. Antibodies were Phycoerythrin (PE)-, PE cy-7-, APC- orBrilliant Violet 421 (BV421)-conjugated, or fluorophore-conjugatedsecondary antibodies were used. Sytox blue was used to exclude deadcells.

Phagocytosis assay. FACS-based phagocytosis assays were performed toevaluate phagocytic abilities of macrophages. Macrophages were harvestedafter 6-8 days of differentiation and divided into FACS tubes orlow-attachment 96-well plates, with 1-5×10⁴ cells per well/tube. Targetcells were added and mixed with macrophages, and incubated at 37° C. for2 hrs with indicated conditions (antibody/drug treatment). For CD47blockade, anti-CD47 (B6H12, BD Biosciences) or humanized anti-CD47(Hu5F9-G4; provided by the CD47 disease team at Stanford University)antibodies were used. Cells were then incubated with PE cy7-conjugatedanti-mouse F4/80 antibody to stain macrophages. After incubation, cellswere washed with FACS buffer and resuspended with FACS buffer containingsytox blue to distinguish dead cells. Phagocytic index was examined byFACS analyses, and macrophages that phagocytosed target cells wereF4/80+ and GFP+. Phagocytic index was calculated with the number ofF4/80+GFP+ cells/divided by the number of F4/80+ cells. In eachexperiment, phagocytic indexes were normalized to the maximal indexes.

Alternatively, macrophages and target cells were mixed and cocultured in24 well plates for 16-24 hrs with indicated conditions. Cells werecollected from the plates by TrypLE and incubated with PE cy7-conjugatedanti-mouse F4/80 antibody to stain for macrophages. After incubation,cells were washed with FACS buffer and resuspended with FACS buffercontaining sytox blue and non-colored standard cells (293T cells wereused as standard cells). Cells were then subjected to FACS analyses.Remaining target cells were normalized to standard cells (numbers ofstandard cells were known and equal in each sample) to evaluate thepercentage of cells phagocytosed by macrophages during the incubation.

For the experiments investigating phagocytic ability of macrophages withdifferential cell surface expression of CRT, cells were stained withanti-F4/80 antibody, as well as anti-calreticulin antibody or isotypecontrol conjugated to the same color after phagocytosis assay. F4/80+cells (macrophages) were gated, among which CRT^(High), CRT^(Medium) orCRT^(Low) cells were analyzed separately for phagocytic indexes.

Overexpression of CRT. Replication-incompetent lentivirus was used tooverexpress calreticulin in J774 cells. CRT cDNA was cloned intopCDH-MCS-IRS-Puro lenti viral vector, with a myc tag after the signalpeptide. The lentiviral vector expressing myc-tagged CRT was transientlytransfected to 293T cells with psPAX2 and pMD2.G at a ratio of (4:3:1).48 hrs after transfection, the supernatant was collected and added toJ774 cells. Cells were treated with puromycin (2 μg/ml) for 48 hrs andselected cells were used for phagocytosis assays. CRT overexpression wasconfirmed by western blot with anti-myc antibody.

Cell surface biotinylation. Mouse bone marrow derived macrophages wereseeded on day 6, and treated with imiquimod or ibrutinib for 16 hrsbefore biotinylation assay. Cells were incubated with NHS-SS-Biotin (0.5mg/ml) for 1 hr in PBS (pH8.0), and then rinsed with quench buffer (20mM Tris-HCl, 120 mM NaCl, pH7.4), 100 mM Glycine in PBS, and PBS. Cellswere lysed in lysis buffer (20 mM Tris-HCl, pH7.4, 150 mM NaCl, 2 mMEDTA, supplemented with 1% Triton X-100, protease inhibitor cocktail andphosphatase inhibitor cocktail). Cell lysate was incubated withneutravidin agarose resin for 4 hr at 4° C., and the resin was washedusing lysis buffer. Biotin-labeled proteins were eluted with lysisbuffer containing 2% SDS and 100 mM DTT, and subjected to SDS-PAGE andimmunoblotting. Intracellular protein GAPDH was used as a negativecontrol to confirm that only cell surface proteins were labeled byNHS-SS-Biotin.

Immunoprecipitation. Calreticulin was immunoprecipitated from J774 cellsexpressing myc-CRT. J774 cells were plated 12 hrs beforeimmunoprecipitation. Cells were treated with imiquimod (1 ug/ml) atindicated time points, and washed with pre-chilled PBS containingphosphatase inhibitors on ice. Cells were then lysed in lysis buffer (20mM Tris-HCl, pH7.4, 150 mM NaCl, 2 mM EDTA, supplemented with 1% TritonX-100, protease inhibitor cocktail and phosphatase inhibitor cocktail).Cell lysate was incubated for 1 hr with GammaBind Plus Sepharose forpreclearance and 4 hr with anti-myc antibodies at 4° C. GammaBindsepharose was added to cell lysate and incubated for 1 hr at 4° C. Beadswere washed with lysis buffer. Proteins were eluted with lysis buffercontaining 2% SDS and 100 mM DTT, and subjected to SDS-PAGE andimmunoblotting. To detect phosphorylated CRT, the blot was incubatedwith biotin-labeled pT66 anti-p-Tyrosine antibody and HRP-conjugatedstreptavidin.

Tumor engraftment and treatment. PC3 cells (human prostate cancer) weresuspended in F-12K medium with 25% matrix matrigel, and injectedsubcutaneously on the back of 6-10 week NSG mice. Mice were treated withPBS or Hu5F9-G4 antibody by intraperitoneal injection 2 weeks afterengraftment, and PBS or TLR agonists (204 of Poly (I:C) HMW and 20 μg ofLPS) by intratumoral injection 7 weeks after engraftment when tumorsreached 100 mm³.

Bioluminescent imaging was performed to monitor tumor growth, asdescribed before. Briefly, D-Luciferin (firefly) potassium salt wasdissolved in PBS to a final concentration of 16.6 mg/ml. Mice wereinjected intraperitoneally with luciferin solution (0.139 g luciferin/kgbody weight), and imaged and analyzed with Living Image 4.0 software.

Tumor dissociation and FACS analyses. Tumor specimens were collectedfrom the mice, minced to pieces smaller than 1 mm in diameter anddissociated in medium 199 with TM enzymes and DNAase at 37° C. untilsingle-cell suspension was achieved. The cells were treated with ACKlysing buffer for lysis of red blood cells, washed twice with HBSS,filtered through 70 μm cell strainer and subjected to flow cytometryanalyses. The cells were stained with anti-CD31 antibody, anti-Gr-1antibody and sytox blue to exclude endothelial cells, neutrophils anddead cells, and with anti-F4/80 antibody for macrophages.

1. A method of increasing phagocytosis of cancer cells, the methodcomprising: contacting a population of phagocytic cells with a TLRagonist in a dose effective to increase expression of calreticulin onthe phagocytic cell surface; and with an effective dose of a CD47blocking agent; wherein programmed cell removal of cancer cells by thephagocytic cells is increased.
 2. The method of claim 1, wherein thecontacting is performed in vivo.
 3. The method of claim 1, wherein thecontacting is performed in vitro.
 4. The method of claim 1, wherein thephagocytic cells are macrophages.
 5. The method of claim 1, wherein theTLR agonist is imiquimod.
 6. The method of claim 1, wherein the TLRagonist is poly I:C.
 7. The method of claim 1, wherein the CD47 blockingagent is an antibody.
 8. The method of claim 7, wherein the antibodyspecifically binds to CD47.
 9. The method of claim 3, wherein thephagocytic cells are introduced into a subject following the contactingwith a TLR agonist and a CD47 blocking agent.
 10. The method of claim 9,wherein expression of calreticulin on the phagocytic cell surface ismeasured prior to the introducing step.
 11. A method of protecting bloodcells from phagocytosis, the method comprising: contacting said cellswith a BTK inhibitory agent.
 12. The method of claim 11, wherein bloodcells in a subject are contacted with a BTK inhibitory agent in vivo.13. The method of claim 12, wherein the subject suffers from amyelodysplastic syndrome.
 14. The method of claim 12, wherein theindividual suffers from autoimmune hemolytic anemia (AIHA).
 15. Themethod of claim 12, wherein the individual suffers from immunethrombocytopenic purpura (ITP)
 16. The method of claim 11, wherein theBTK inhibitory agent is ibrutinib.